Numerous methods used to detect and characterize nucleic acid structures employ tagging schemes that rely on electromagnetic radiation (EM) emission of an excited state light-absorbing chromophore. Examples of such photoluminescent processes include phosphorescence and fluorescence emission. Fluorescence detection, for example, has been used in DNA sequencing to great effect due, in part, to the high degree of sensitivity allowing single molecule detection.
Performing iterative fluorescent detection steps in an array context, such as sequencing by synthesis, can cause fluorescence signal intensity loss (see, for example, Fedurco et al. WO2006/064199). This problem was addressed, in part, by the addition of ascorbate to a detection solution to increase the number of useful detection cycles from about eight to ten cycles, in the absence of ascorbate, to about 25 cycles in the presence of ascorbate. The possible mechanisms that underlie this signal loss are numerous, and can include cleavage of individual nucleic acid members from the support.
There are a number of pathways by which nucleic acid damage can occur during irradiation in fluorescence detection. Fluorescence emission normally occurs with the emission of light of a longer wavelength (lower energy), than the original irradiating source. However, under conditions in which intense EM radiation is being absorbed by the fluorophore, such as in laser-induced fluorescence (LIF), it is possible for a molecule to absorb two photons, which can lead to the emission of higher energy radiation of smaller wavelengths than the original excitation source. This multiple photon absorption can cause the fluorophore to emit EM radiation in the UV-visible region which can contribute to nucleic acid base dimerization and/or the generation of reactive oxygen species.
For example, it has been indicated that exposure of whole cells to ultraviolet (UV) radiation can cause DNA damage via the direct photochemical [2+2] photocycloaddition reaction of thymine or cytosine to provide cyclobutane pyrimidine dimers, such as TT, TC, and CC. Such direct photocycloaddition reactions can occur in the UV B and UV C regions which extend from about 100 nm to about 315 nm.
In the UV A region through a portion of the visible region, spanning from about 315 nm to about 500 nm, a complex mixture of indirect mechanisms can also cause DNA damage through photosensitization of other cellular components. Such indirect mechanisms can result in pyrimidine dimer formation and oxidative DNA modification via reactive species such as singlet oxygen, superoxide anion, and iron-promoted hydroxyl radical formation. Finally, it also has also been indicated that reactive singlet oxygen can be generated by fluorescence quenching of an excited state fluorophore by triplet oxygen. Any combination of direct or indirect pyrimidine dimerization and nucleic acid damage due to various reactive oxygen species observed in whole cells can be the underlying cause of fluorescence signal intensity loss observed in the array context.
There is a need to further reduce fluorescent signal intensity loss for applications in sequencing by synthesis to facilitate sequencing of long nucleotide sequences, including sequences of 50, 75, 100, 200, and 500 nucleotides or more. Moreover, solutions to fluorescent signal intensity loss in the context sequencing by synthesis are readily applicable to other nucleic acid detection platforms that employ multiple irradiation steps. The present invention satisfies this need and provides related advantages as well.